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Equine Veterinary Journal ISSN 0425-1644 DOI: 10.1111/evj.12103
Editorials
Rhodococcus equi research 2008–2012: Report of the Fifth International Havemeyer Workshop Introduction Rhodococcus equi pneumonia in foals is a continued cause of concern to the equine industry owing to its high incidence and considerable economic impact on stud farms worldwide [1]. Seventy scientists from 14 countries met at the Villa Le Cercle in Deauville (France) on 9–12 July 2012 as part of the 5th International Havemeyer Workshop on Rhodococcus equi to discuss recent developments and research progress since the last meeting in Edinburgh 2008 [2]. Understanding rhodococcal pneumonia requires an integrated appraisal of the pathogen–host–environment interplay, and the components of this triad were dissected and examined in the sessions of the workshop.
Rhodococcus equi biology and virulence The conference was opened by José Vázquez-Boland (University of Edinburgh, UK) with an introductory lecture that illustrated the impact of the R. equi genome project [3] in advancing research into R. equi pathogenesis and vaccinology. The Edinburgh group found that R. equi produces pili appendages and that these are a key virulence factor mediating attachment to host cells. Using a novel murine model of R. equi infection (presented by Mariela Scortti), it was shown that the R. equi pili (Rpl) are essential for lung colonisation in vivo and elicit full protection in vaccinated mice. The Rpl pili are a major immunodominant antigen in foals and may form the basis of a protein vaccine to prevent rhodococcal pneumonia during the early stages of infection and a serodiagnostic test to detect infected animals. Analysis of the genome has identified additional chromosomal virulence factors. Collaborative work between Edinburgh and Wim Meijer’s group at University College Dublin (Ireland) focused on the nutritional and metabolic determinants of rhodococcal pathogenesis. Alexia Hapeshi (Edinburgh) reported on a vap pathogenicity island (PAI)coexpressed glutamate synthase enzyme that is required for growth in low nitrogen and is essential for R. equi virulence; Aleksandra MirandaCasoLuengo (Dublin) and Héctor Rodríguez/Alexia Hapeshi (Edinburgh) reported that lactate and urea are potential host-derived carbon and nitrogen sources for the intracellular growth of R. equi in macrophages; and Raúl Miranda-CasoLuengo (Dublin) reported that R. equi scavenges iron in vivo via the virulence-associated siderophore Rhequichelin, a study carried out in collaboration with Mary Hondalus’ group (University of Georgia, USA) [4]. Additional potential chromosomal virulence factors that were discussed include the polysaccharide capsule (Iain MacArthur, Edinburgh) and catalases [5] (Pauline Bidaud, Equine Diseases Laboratory, ANSES, Dozulé, France). Albert Haas (University of Bonn, Germany) presented an overview of his research on the cell biology of R. equi infection. Like other intracellular parasites of macrophages, R. equi subverts phagosome trafficking to create a hospitable membrane-bound vacuole, in which the pathogen can proliferate. New data from this group suggest a key role for the late endosomal GTPase Rab7, a molecular switch that controls phagolysosome biogenesis, in modulating the intracellular proliferation of R. equi. In the absence of Rab7 and thus of late endosome-lysosome fusion, R. equi bacteria can proliferate within macrophages without the need for the virulence plasmid, reinforcing the notion that the plasmid supports intracellular survival by preventing lysosomal killing. The virulence plasmid remains a major focus of interest since the seminal work in the 1990s by the groups of Shinji Takai (Kitasato University, Japan) and John Prescott (University of Guelph, Ontario, Canada) identified this extrachromosomal element as essential for R. equi intramacrophage survival and pathogenicity [6,7]. Mary Hondalus presented interesting new data on the conjugal transfer properties of the R. equi virulence plasmid. This plasmid can easily be lost in the absence Equine Veterinary Journal 45 (2013) 523–526 © 2013 EVJ Ltd
of host selective pressure (e.g. upon repeated subculture in vitro) and plasmidless/avirulent strains are abundant in soil. The Hondalus’ group showed that avirulent strains can potentially regain the plasmid (and thus virulence) by conjugation at a high frequency of up to 10–1 conjugants per recipient with donor virulent bacteria [8]. This observation has important implications for our understanding of R. equi epidemiology because it implies that virulent isolates from subclinically infected animals can rapidly propagate the virulent phenotype among resident nonvirulent R. equi bacteria present in the farm environment. A significant emerging aspect is the host tropism conferred by the virulence plasmid (reports by Ana Valero-Rello, Edinburgh, and Jennifer Willingham-Lane, University of Georgia, USA). Earlier work indicated the existence of different host-associated R. equi virulence plasmids, each encoding specific virulence-associated protein (Vap) variants, namely the equine pVAPA, porcine pVAPB and a third type associated with bovine isolates [9]. The Edinburgh group has now sequenced the bovine virulence plasmid, designated pVAPN. Interestingly, it is a large linear replicon unrelated to the circular equine and porcine plasmids but similar to other rhodococcal linear replicons. Mechanistic analysis of the plasmid vap PAI has been undertaken by Wim Meijer’s group, with detailed analysis on the regulation of the vap genes and protein–protein interaction studies between the different Vap proteins. The latter appear to form a multisubunit complex, the significance of which is currently under investigation. Wim Meijer also presented a novel, real-time, impedance-based method to assess R. equi virulence, which allows high-throughput screening of mutants [10]. The virulence plasmid was also found to support intracellular proliferation of R. equi in Acanthamoeba, suggesting that bacteriovorous protists could have been training grounds for the evolution of the virulence plasmid and may act as environmental reservoirs for virulent R. equi (presentation by Mariela Scortti, Edinburgh). Iain Sutcliffe (Northumbria University, UK) presented a study based on molecular typing and numerical taxonomy methods that proposed the reclassification of R. equi into a novel genus, Prescottia, with P. equi as its only species. In the following debate it was noted that this proposal clashed with previous comparative genomics studies [3] and other lines of genetic evidence (such as the sharing with other rhodococci of the same plasmid-driven niche-adaptive strategy, based on related circular and linear replicons) indicating that R. equi is well accommodated within the current genus Rhodococcus. Concerns were raised that changing a well-consolidated name in veterinary, medical and scientific/technical literature may cause confusion and would be justifiable only if based on comprehensive re-examination of the entire genus Rhodococcus using modern phylogenomics approaches.
Clinical aspects Early detection of subclinically infected foals using thoracic ultrasonography has been pursued in recent years as a strategy for early diagnosis of rhodococcal pneumonia and targeting the application of antimicrobial treatments on endemic farms. Two field studies, by Monica Venner (Destedt Veterinary Clinic, Germany) and Keith Chaffin (Texas A&M University, USA), assessed the validity of the ultrasonographic screening approach. Both studies showed that the majority of foals with subclinical pulmonary abscesses compatible with rhodococcal infection recover spontaneously without progressing to clinically apparent R. equi pneumonia. The Venner study included a placebo-controlled comparison of different combinations of antimicrobials currently used in foals showing ultrasonographic consolidations. No differences were observed between treated and untreated groups in recovery or progression to clinical pneumonia. These findings have important implications, because they
523
Rhodococcus equi research 2008–2012: 5th International Havemeyer Workshop
clearly question the usefulness of systematic ultrasonographic screening programmes and the mass antimicrobial treatment of foals [11]. Antimicrobial prophylaxis against rhodococcal pneumonia becomes even more questionable in light of the findings of a study presented by Steeve Giguère (University of Georgia, USA) and other US colleagues, reporting the emergence of extensive macrolide and rifampin resistance among R. equi isolates on a farm where widespread use of these drugs was instituted to control pulmonary lesions identified ultrasonographically [12]. As commonly seen in soil-dwelling bacteria, R. equi is resistant to many antimicrobials due to the presence of numerous chromosomal resistance determinants [2]. The efficacy of antimicrobial drugs and dosing regimens to control R. equi is thus under continual scrutiny. Londa Berghaus (University of Georgia) reported on the in vitro interactions, pharmacodynamics, mutant prevention concentration and post antibiotic effect of 11 antimicrobial agents against R. equi [13]. Macrolides and either rifampin or doxycycline were synergistic, whereas aminoglycosides and macrolide or rifampin combinations were antagonistic. Jean-Marie Bryskier (Veterinary School of Alfort, France) reported on the in vitro activity of ceftiofur and marbofloxacin against R. equi and also showed that both drugs inhibit the intracellular growth of R. equi in macrophages. Monica Venner also presented data indicating that co-administration of clarithromycin with rifampin significantly decreases concentrations of clarithromycin and possibly other macrolides in plasma and in the lungs. The limited number of effective drugs and the emergence of resistance make it necessary to explore alternative approaches. Margot Schlusselhuber (Equine Diseases Laboratory, ANSES) presented data on the efficacy of equine antimicrobial peptide eCATH1. The peptide was found to kill R. equi and other common bacterial pathogens, such as Klebsiella pneumoniae and Streptococcus equi subspecies zooepidemicus, at micromolar concentrations that were not cytotoxic to mammalian cells [14], and to be active inside macrophages and synergistic with rifampin in both ex vivo and in vivo experiments. Two presentations on rhodococcal pneumonia in Sweden and Hokkaido district in Japan, both characterised by a humid climate with icy winters and relatively cool summers, help dissipate the myth that R. equi infection is restricted to warmer countries with dry summers. The first study, presented by Gittan Gröndahl (National Veterinary Institute, Uppsala, Sweden), reported 91 cases of R. equi pneumonia diagnosed in 2010, with 21% showing extrapulmonary manifestations and an overall case-fatality rate of 19%. Shinji Takai’s group (Veterinary School, Kitasato University, Japan) found a seasonal correlation for tracheal colonisation by R. equi on endemic farms, with a constant monthly increase of positivity (from 80%) and in the average number of bacteria in the aspirate in foals born in the period from February to May. It is unclear to what extent this is due to purely environmental factors (i.e. build-up in soil) or epidemiological factors (i.e. accumulation of carrier animals, hence increased transmission as the foaling season progresses). Another study of tracheobronchial aspirates, presented by S. Giguère, showed that the presence of mixed cultures of R. equi in association with other Gram-positives, Gram-negatives or fungi is not necessarily associated with worse prognosis. These mixed cultures were significantly more likely to be encountered in tracheobronchial aspirates than in lung tissue, reminding us that many organisms may colonise the trachea without necessarily contributing to pulmonary pathology [15]. While mostly seen in foals
Equine Veterinary Journal ISSN 0425-1644 DOI: 10.1111/evj.12103
Editorials
Rhodococcus equi research 2008–2012: Report of the Fifth International Havemeyer Workshop Introduction Rhodococcus equi pneumonia in foals is a continued cause of concern to the equine industry owing to its high incidence and considerable economic impact on stud farms worldwide [1]. Seventy scientists from 14 countries met at the Villa Le Cercle in Deauville (France) on 9–12 July 2012 as part of the 5th International Havemeyer Workshop on Rhodococcus equi to discuss recent developments and research progress since the last meeting in Edinburgh 2008 [2]. Understanding rhodococcal pneumonia requires an integrated appraisal of the pathogen–host–environment interplay, and the components of this triad were dissected and examined in the sessions of the workshop.
Rhodococcus equi biology and virulence The conference was opened by José Vázquez-Boland (University of Edinburgh, UK) with an introductory lecture that illustrated the impact of the R. equi genome project [3] in advancing research into R. equi pathogenesis and vaccinology. The Edinburgh group found that R. equi produces pili appendages and that these are a key virulence factor mediating attachment to host cells. Using a novel murine model of R. equi infection (presented by Mariela Scortti), it was shown that the R. equi pili (Rpl) are essential for lung colonisation in vivo and elicit full protection in vaccinated mice. The Rpl pili are a major immunodominant antigen in foals and may form the basis of a protein vaccine to prevent rhodococcal pneumonia during the early stages of infection and a serodiagnostic test to detect infected animals. Analysis of the genome has identified additional chromosomal virulence factors. Collaborative work between Edinburgh and Wim Meijer’s group at University College Dublin (Ireland) focused on the nutritional and metabolic determinants of rhodococcal pathogenesis. Alexia Hapeshi (Edinburgh) reported on a vap pathogenicity island (PAI)coexpressed glutamate synthase enzyme that is required for growth in low nitrogen and is essential for R. equi virulence; Aleksandra MirandaCasoLuengo (Dublin) and Héctor Rodríguez/Alexia Hapeshi (Edinburgh) reported that lactate and urea are potential host-derived carbon and nitrogen sources for the intracellular growth of R. equi in macrophages; and Raúl Miranda-CasoLuengo (Dublin) reported that R. equi scavenges iron in vivo via the virulence-associated siderophore Rhequichelin, a study carried out in collaboration with Mary Hondalus’ group (University of Georgia, USA) [4]. Additional potential chromosomal virulence factors that were discussed include the polysaccharide capsule (Iain MacArthur, Edinburgh) and catalases [5] (Pauline Bidaud, Equine Diseases Laboratory, ANSES, Dozulé, France). Albert Haas (University of Bonn, Germany) presented an overview of his research on the cell biology of R. equi infection. Like other intracellular parasites of macrophages, R. equi subverts phagosome trafficking to create a hospitable membrane-bound vacuole, in which the pathogen can proliferate. New data from this group suggest a key role for the late endosomal GTPase Rab7, a molecular switch that controls phagolysosome biogenesis, in modulating the intracellular proliferation of R. equi. In the absence of Rab7 and thus of late endosome-lysosome fusion, R. equi bacteria can proliferate within macrophages without the need for the virulence plasmid, reinforcing the notion that the plasmid supports intracellular survival by preventing lysosomal killing. The virulence plasmid remains a major focus of interest since the seminal work in the 1990s by the groups of Shinji Takai (Kitasato University, Japan) and John Prescott (University of Guelph, Ontario, Canada) identified this extrachromosomal element as essential for R. equi intramacrophage survival and pathogenicity [6,7]. Mary Hondalus presented interesting new data on the conjugal transfer properties of the R. equi virulence plasmid. This plasmid can easily be lost in the absence Equine Veterinary Journal 45 (2013) 523–526 © 2013 EVJ Ltd
of host selective pressure (e.g. upon repeated subculture in vitro) and plasmidless/avirulent strains are abundant in soil. The Hondalus’ group showed that avirulent strains can potentially regain the plasmid (and thus virulence) by conjugation at a high frequency of up to 10–1 conjugants per recipient with donor virulent bacteria [8]. This observation has important implications for our understanding of R. equi epidemiology because it implies that virulent isolates from subclinically infected animals can rapidly propagate the virulent phenotype among resident nonvirulent R. equi bacteria present in the farm environment. A significant emerging aspect is the host tropism conferred by the virulence plasmid (reports by Ana Valero-Rello, Edinburgh, and Jennifer Willingham-Lane, University of Georgia, USA). Earlier work indicated the existence of different host-associated R. equi virulence plasmids, each encoding specific virulence-associated protein (Vap) variants, namely the equine pVAPA, porcine pVAPB and a third type associated with bovine isolates [9]. The Edinburgh group has now sequenced the bovine virulence plasmid, designated pVAPN. Interestingly, it is a large linear replicon unrelated to the circular equine and porcine plasmids but similar to other rhodococcal linear replicons. Mechanistic analysis of the plasmid vap PAI has been undertaken by Wim Meijer’s group, with detailed analysis on the regulation of the vap genes and protein–protein interaction studies between the different Vap proteins. The latter appear to form a multisubunit complex, the significance of which is currently under investigation. Wim Meijer also presented a novel, real-time, impedance-based method to assess R. equi virulence, which allows high-throughput screening of mutants [10]. The virulence plasmid was also found to support intracellular proliferation of R. equi in Acanthamoeba, suggesting that bacteriovorous protists could have been training grounds for the evolution of the virulence plasmid and may act as environmental reservoirs for virulent R. equi (presentation by Mariela Scortti, Edinburgh). Iain Sutcliffe (Northumbria University, UK) presented a study based on molecular typing and numerical taxonomy methods that proposed the reclassification of R. equi into a novel genus, Prescottia, with P. equi as its only species. In the following debate it was noted that this proposal clashed with previous comparative genomics studies [3] and other lines of genetic evidence (such as the sharing with other rhodococci of the same plasmid-driven niche-adaptive strategy, based on related circular and linear replicons) indicating that R. equi is well accommodated within the current genus Rhodococcus. Concerns were raised that changing a well-consolidated name in veterinary, medical and scientific/technical literature may cause confusion and would be justifiable only if based on comprehensive re-examination of the entire genus Rhodococcus using modern phylogenomics approaches.
Clinical aspects Early detection of subclinically infected foals using thoracic ultrasonography has been pursued in recent years as a strategy for early diagnosis of rhodococcal pneumonia and targeting the application of antimicrobial treatments on endemic farms. Two field studies, by Monica Venner (Destedt Veterinary Clinic, Germany) and Keith Chaffin (Texas A&M University, USA), assessed the validity of the ultrasonographic screening approach. Both studies showed that the majority of foals with subclinical pulmonary abscesses compatible with rhodococcal infection recover spontaneously without progressing to clinically apparent R. equi pneumonia. The Venner study included a placebo-controlled comparison of different combinations of antimicrobials currently used in foals showing ultrasonographic consolidations. No differences were observed between treated and untreated groups in recovery or progression to clinical pneumonia. These findings have important implications, because they
523
Rhodococcus equi research 2008–2012: 5th International Havemeyer Workshop
clearly question the usefulness of systematic ultrasonographic screening programmes and the mass antimicrobial treatment of foals [11]. Antimicrobial prophylaxis against rhodococcal pneumonia becomes even more questionable in light of the findings of a study presented by Steeve Giguère (University of Georgia, USA) and other US colleagues, reporting the emergence of extensive macrolide and rifampin resistance among R. equi isolates on a farm where widespread use of these drugs was instituted to control pulmonary lesions identified ultrasonographically [12]. As commonly seen in soil-dwelling bacteria, R. equi is resistant to many antimicrobials due to the presence of numerous chromosomal resistance determinants [2]. The efficacy of antimicrobial drugs and dosing regimens to control R. equi is thus under continual scrutiny. Londa Berghaus (University of Georgia) reported on the in vitro interactions, pharmacodynamics, mutant prevention concentration and post antibiotic effect of 11 antimicrobial agents against R. equi [13]. Macrolides and either rifampin or doxycycline were synergistic, whereas aminoglycosides and macrolide or rifampin combinations were antagonistic. Jean-Marie Bryskier (Veterinary School of Alfort, France) reported on the in vitro activity of ceftiofur and marbofloxacin against R. equi and also showed that both drugs inhibit the intracellular growth of R. equi in macrophages. Monica Venner also presented data indicating that co-administration of clarithromycin with rifampin significantly decreases concentrations of clarithromycin and possibly other macrolides in plasma and in the lungs. The limited number of effective drugs and the emergence of resistance make it necessary to explore alternative approaches. Margot Schlusselhuber (Equine Diseases Laboratory, ANSES) presented data on the efficacy of equine antimicrobial peptide eCATH1. The peptide was found to kill R. equi and other common bacterial pathogens, such as Klebsiella pneumoniae and Streptococcus equi subspecies zooepidemicus, at micromolar concentrations that were not cytotoxic to mammalian cells [14], and to be active inside macrophages and synergistic with rifampin in both ex vivo and in vivo experiments. Two presentations on rhodococcal pneumonia in Sweden and Hokkaido district in Japan, both characterised by a humid climate with icy winters and relatively cool summers, help dissipate the myth that R. equi infection is restricted to warmer countries with dry summers. The first study, presented by Gittan Gröndahl (National Veterinary Institute, Uppsala, Sweden), reported 91 cases of R. equi pneumonia diagnosed in 2010, with 21% showing extrapulmonary manifestations and an overall case-fatality rate of 19%. Shinji Takai’s group (Veterinary School, Kitasato University, Japan) found a seasonal correlation for tracheal colonisation by R. equi on endemic farms, with a constant monthly increase of positivity (from 80%) and in the average number of bacteria in the aspirate in foals born in the period from February to May. It is unclear to what extent this is due to purely environmental factors (i.e. build-up in soil) or epidemiological factors (i.e. accumulation of carrier animals, hence increased transmission as the foaling season progresses). Another study of tracheobronchial aspirates, presented by S. Giguère, showed that the presence of mixed cultures of R. equi in association with other Gram-positives, Gram-negatives or fungi is not necessarily associated with worse prognosis. These mixed cultures were significantly more likely to be encountered in tracheobronchial aspirates than in lung tissue, reminding us that many organisms may colonise the trachea without necessarily contributing to pulmonary pathology [15]. While mostly seen in foals
